CN102203246A - Controllable assembly and disassembly of nanoparticle systems via protein and DNA agents - Google Patents

Controllable assembly and disassembly of nanoparticle systems via protein and DNA agents Download PDF

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CN102203246A
CN102203246A CN2009801297436A CN200980129743A CN102203246A CN 102203246 A CN102203246 A CN 102203246A CN 2009801297436 A CN2009801297436 A CN 2009801297436A CN 200980129743 A CN200980129743 A CN 200980129743A CN 102203246 A CN102203246 A CN 102203246A
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nano particle
ssdna
dna
assembling
rna
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李树官
奥列格·甘格
丹尼尔·范德勒利耶
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Brookhaven Science Associates LLC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles

Abstract

The invention relates to the use of peptides, proteins, and other oligomers to provide a means by which normally quenched nanoparticle fluorescence may be recovered upon detection of a target molecule. Further, the inventive technology provides a structure and method to carry out detection of target molecules without the need to label the target molecules before detection. In another aspect, a method for forming arbitrarily shaped two- and three- dimensional DNA binding protein-mediated nanoparticle structures and the resulting structures are described. Specifically embodied is the use of the gene 5 protein (g5p). Proteins mediating structure formation may themselves be functionalized with a variety of useful moieties, including catalytic functional groups.

Description

Conciliate the nanoparticle system of assembling by protein and the controlled assembling of DNA agent
The application is international (PCT) application, and it requires the U.S. Provisional Application submitted on June 2nd, 2008 number is 61/058,037 right of priority, and it is incorporated in herein with way of reference.
The present invention is subsidized by USDOE, makes under government supports with contract number DE-AC02-98CH10886.Government has certain right to the present invention.
Technical field
In case a kind of method or device that the nano-particle fluorescence that the present invention relates to utilize peptide, protein and other oligomer to provide and detect the normal quencher of target molecule may be resumed.In addition, the invention provides structure and the method that the labels targets molecule can carry out the target molecule detection before detecting that need not.
Background technology
Nanotechnology research and result thereof have been used for many different research fields, and are applied to scientific research and industrial technology, as nano electron device, transmitter and catalyzer.Be in the energy, medical treatment of frontier and the relevant nanotechnology of safety and will depend on integration and optimization based on nano particle technology and bio-science, thereby be designed for more and more mixing (hybridization) material of cleaning and energy transformation and storage more efficiently, and biosensor with stronger sensitivity.Such integration not only needs accurately to control nanoparticle size, shape, composition and surface property, but also need be with the ability of controlled kinetics with the final assembling morphology self-assembly and the nano particle that dissociates under the condition that living things system is allowed.
At present, utilize biological member (building blocks) self-assembly to have metal (Au, Ag, Pt), semi-conductor (CdSe, CdS, ZnS, GaAs) and magnetic (Fe 2O 3) nano particle of characteristic realizes by two kinds of main paties: (i) based on the system of DNA with (ii) based on the system of protein-(peptide-).The remarkable specificity of DNA and programmable interaction allow the self-assembly of DNA bonded nano particle and the structure of complex construction.Their complicacy and function can be expanded by the protein of having incorporated the biosensor effect into or having played organizer (organizer) effect of complicated support.Shown very big potential based on the self-assembly system of DNA as biosensor.Proved that peptide combines with the specificity of inorganic surfaces, and by utilizing the phage display system can select these peptides.And this peptide has been successfully used to the self-assembly of the structure and the inorganic nanoparticles of nanostructure.Because proteinic functional diversity, mix (hybridization) nano material aspect and receive sizable concern based on the design functionality that is combined in of DNA and proteinic system.
Utilize the self-assembly and the programmable complex construction of the nano particle of DNA to be proved to be.The self-assembly of DNA inductive nano particle at first 1996 by Mirkin and colleague at it " ADNA-based method for rationally assembling nanoparticles into macroscopicmaterials; " Nature, 382 (6592): p.607-609,1996, with Alivisatos and colleague thereof, " Organization of ' nanocrystal molecules ' using DNA; " Nature, 382 (6592): p.609-611, introduced in 1996 the initiative paper, these two pieces of articles all by reference integral body be incorporated in this.The specific hybrid ability of DNA is used for the self-assembly of nano particle, on nano particle chemical fixation single stranded DNA (ssDNA).Gathering is by adding and being attached to described particulate ssDNA complementary strand linker DNA acquisition.The gathering of ssDNA bonded nano particle is attended by physics and change of optical property, and is used to detect DNA.Adding with joint complementary dna fragmentation causes aggregation to dissociate.Replacedly, this system can be designed to dna fragmentation to be detected and serves as this joint.Depend on the feasible self-assembly (Fig. 1) that can reverse aggregation of DNA pre-arcing characterisitics of sequence and length by the raising temperature.The sequence dependent pre-arcing characterisitics allows to utilize ssDNA bonded nano particle to detect simple point mutation.
Hold complementary fluorescence oligonucleotide to be used as molecular beacon thereby be designed for, be used to detect the target DNA of hybridizing, cause the fluorescent emission of quencher with this hairpin structure in conjunction with the gold nano grain quencher that forms hairpin structure 3 ' and 5 '.
Microarray technology is used for high-throughput routinely and quantizes a large amount of different DNA or RNA fragments.System based on array need be by complex sign target DNA or the RNA of C-DNA.SsDNA-bonded nano particle has been used as the material standed for of the target molecule of surveying them.
In addition, the specificity of the DNA assembly of the member be used for creating DNA member and complex structure geometry and form that interacts.
Summary of the invention
Though above-mentioned system needs the prerequisite of the hybridization of complementary dna chain as correct assembling, and realizes separating assembling (disassembly) by melting DNA at elevated temperatures, this process will cause funtion part usually, as proteinic irreversible deactivation.Method of the present invention allows the bonded nano particle of controlled assembling incomplementarity single stranded DNA-(ssDNA-), and it utilizes gene 5 protein (g5p) to be attached to two antiparallel ssDNA chains as molecule " cementing agent (glue) ".The dynamic (dynamical) control of the assembling of particle aggregate can obtain by hybridizing with the sequence-specific of complementary ssDNA (C-ssDNA), and the size of aggregate is controlled by the concentration of regulating g5p.The controlled assembling of separating of g5p-ssDNA mixture can cause by the hybridization with C-ssDNA, and it allows to regulate assembling kinetics and particle aggregate effectively decomposes under room temperature or physiological temp.In addition, g5p albumen allows the constructing function recombinant derivative, and this derivative contains polyhistidyl or allows these protein to be attached to other affinity tag of nano grain surface specifically.This allows to need not thermal treatment is that assembling is conciliate in the assembling of may command nano particle, and easily protein is incorporated in the nanostructure based on DNA, gives the potential that is designed to composite nano materials.
In case method or device that the nano-particle fluorescence that the present invention relates to utilize peptide, protein and other oligomer to provide and detect the normal quencher of target molecule may be resumed.In addition, technology of the present invention provides structure and the method that the labels targets molecule can carry out the detection of target molecule before detection that need not.On the other hand, the method for nanoparticle structure of the two and three dimensions protein mediation that is used to form arbitrary shape and the structure of gained have been described.The structure of protein mediation forms self can utilize multiple useful part to come functionalization.
In some embodiments of the present invention, provide and utilized g5p albumen or the protein-bonded controlled and reversible nanoparticle assemblies of similar DNA.In some modification, this nano particle is wrapped (encapsulated) in DNA and/or RNA, and this nano particle and nucleotide binding protein assembling.In other modification, this nano particle is wrapped up by nucleotide binding protein, and this nano particle and DNA and/or RNA assembling.Some embodiments provide multidimensional structure, and it comprises branch, nucleotide binding protein and the nano particle of single stranded DNA and/or RNA.Some embodiments of the present invention also provide method or the technology of utilizing nucleotide binding protein to prepare the controlled and reversible assembly of nano particle.
Other embodiment of the present invention provides the DNA assembly (assemblage) of nucleic acid binding protein mediation, and it comprises incomplementarity DNA or the RNA chain that is attached on the nucleotide binding protein.
Other embodiment of the present invention provides molecular switch, and it comprises " shutoff " position of quenching of fluorescence and " unlatching " position of fluorescent emission, comprises ssDNA that is attached on the fluorescein and the 2nd ssDNA that is attached on the DABCYL.This switch can be by making the hybridization of complementary ssDNA or ssRNA and fluorescein-ssDNA chain or ssDNA-DABCYL chain by " unlatching ".
Description of drawings
Fig. 1 shows the self-assembly of ssDNA-bonded nano particle and conciliates the art methods of assembling.
Fig. 2 illustrates the assembling of controlled g5p-mediation and conciliates package technique.
Fig. 3 illustrates the assembling of the g5p bonded nano particle of ssDNA mediation and conciliates the synoptic diagram of assembling.
Fig. 4 shows the operation of the molecular switch of g5p-mediation.
Fig. 5 shows the self-assembly of orderly peptide bonded g5p and DNA nano particle.
Fig. 6 A and Fig. 6 B show the result that electrophoretic mobility is measured.
Fig. 7 A, 7B and 7C are the assembling data from the ssDNA-bonded gold grain of g5p-mediation.
Fig. 8 A and 8B show the dynamic light scattering of ssDNA-bonded gold grain and the result that ultraviolet-visible is analyzed.
Fig. 9 A and 9B show the synoptic diagram of the protein mediated ssDNA-bonded gold grain assembling of g5p respectively, and the variation of the hydromeehanics diameter value of such assembling.
Figure 10 A, 10B and 10C illustrate the decomposition of the ssDNA-bonded gold grain aggregate of g5p-mediation.
Figure 11 shows the influence of the adding of C-ssDNA to the gold grain aggregate.
Embodiment
Opposite with DNA, protein not only has the specific combination characteristic, but also has abundant catalysis characteristic.The interaction of protein and peptide has been used for the self-assembly and the ordering (ordering) of nano particle.There is specific peptide to utilize peptide library to select to mineral compound (metal, semi-conductor and magnetic compound), and successfully is used for the self-assembly and the biomineralization of inorganic nanoparticles.Notice the specific interaction between peptide and the inorganic surfaces, thereby the inventor thinks that inorganic nanoparticles can be connected show peptide with protein by proteinic genetically engineered.
Combination based on DNA and proteinic system is noticeable as the application of biosensor or complicated support organizer because of having expanded them.Vitamin H bonded DNA can make the member that is used as assembling on the crosslinked Streptavidin of this vitamin H bonded DNA by being attached to.Antibody has been used for the self-assembly of the auxiliary DNA-bonded of protein nano particle, and this nano particle contains the target molecule of antibody.
Although have these progress aspect the nanometer materials, still have many restrictions and technology barrier utilizing DNA and protein to make up.DNA need be used to expect the suitable condition of hybridizing.There is inherent limitations in DNA hybridization, as G-C content, salt concn and temperature.When homogeneous, only by the kinetics and the size of control assembly, the protein of forming in the structure distributes the permission entire structure to have identical functionality.During the self-assembly of the ssDNA of DNA-hybridized induction bonded nano particle, be difficult in not chemically modified oligonucleotide, for example, introduce protein under the biotinylated situation, thus conjugated protein.The temperature that increases can be induced biomolecules, especially proteinic irreversible deactivation.Separating assembling and temperature need being brought up to more than the melting temperature (Tm) of the aggregate that is formed by DNA hybridization is generally about more than 55 ℃, and it can cause the restriction that is ready to use in the system in the coenocorrelation.To detect target molecule based on the system of array (DNA, RNA), mark is crucial, expensive and step consuming time in order to utilize.The mark of target molecule needs the other time to be used for specimen preparation, restriction rapid detection or diagnostic biomolecule.
Utilize gene 5 protein (g5p) as molecule " cementing agent ", overcome by many restrictions in conjunction with DNA and above-mentioned protein manufacturing nanometer materials in conjunction with two antiparallel incomplementarity single stranded DNAs (ssDNA).Also can utilize the other hybridization with complementary ssDNA (C-ssDNA), it causes the g5p-ssDNA complex dissociation.
One aspect of the present invention provides the assembly and its method of preparation of controlled and reversible nano particle.
In some embodiments of the present invention, this nano particle is a metal.In preferred embodiment, this metal is gold and silver or platinum.In preferred embodiment, this nano particle is a gold.In other embodiments, this nano particle is a semi-conductor.In some modification, this semi-conductor is cadmium selenide, Cadmium Sulfide, zinc sulphide, or gallium arsenide.In other embodiments, this nano particle is a magnetic.In some modification, this magnetic nanoparticle comprises ferriferous oxide.
In some embodiments of nanoparticle assemblies, this DNA and/or RNA are strand or two strands.In preferred embodiment, this DNA is a strand.
In some embodiments, this nucleotide binding protein is any protein that can be incorporated into DNA or RNA.In preferred embodiment, this nucleotide binding protein is gene 5 albumen (g5p).
In some embodiments, provide use g5p albumen or similar DNA protein-bonded controlled and reversible nanoparticle assemblies.
In some embodiments, this controlled and reversible nanoparticle assemblies comprises the nano particle with incomplementarity DNA and/or RNA parcel.In preferred embodiment, the nano particle that wraps up with incomplementarity DNA and/or RNA combines by nucleotide binding protein, thereby forms nanoparticle assemblies.By this way, formed controlled and the reversible nanoparticle assemblies.
In the other embodiment of controlled and reversible nanoparticle assemblies, make this nanoparticle assemblies separate assembling by further making this nanoparticle assemblies and combining with incomplementarity DNA and/or RNA complementary DNA and/or RNA.By this way, realization is controlled separates assembling with the reversible nano particle.
Method of the present invention provides utilizes g5p albumen or the protein-bonded controlled incomplementarity ssDNA-bonded nanoparticle assemblies of similar DNA.Can be used to regulate assembling kinetics in assembling stage and lower concentration C-ssDNA hybridization.In addition, under room temperature (Fig. 2) or physiological temp, utilized C-ssDNA to realize effective decomposition of particle aggregate.This approach makes that need not thermal treatment is that assembling is conciliate in the assembling of may command nano particle, and the mixing based on g5p (hybridization) albumen that for example contains affinity tag or other catalyst structure domain is incorporated in the nanostructure based on DNA, give the potential of design composite nano materials.
In some embodiments of this method, the assembling of controllable nano particle is conciliate assembling process and is prepared as follows: with DNA and/or RNA parcel nano particle, the nano particle of parcel is combined with nucleotide binding protein, and this DNA and/or RNA are attached on this nucleotide binding protein.By this way, prepared controllable nano groups of grains process of assembling.
In some modification of this method,, make the assembly of this nano particle separate assembling by further making this nanoparticle assemblies and combining with incomplementarity DNA and/or RNA complementary Nucleotide.By this way, the controlled nano particle of realization is separated assembling process.
In some embodiments of this method, nanoparticle assemblies prepares by the following: with incomplementarity ssDNA functionalization a plurality of nano particles, the nano particle of this functionalization is exposed to gene 5 albumen, and at least two chains of ssDNA are linked to each other with g5p.
Conciliate assembling in order to obtain nano particle in response to the assembling of the further sensitivity of target DNA, the present invention has considered use g5p bonded nano particle, and it can be by incomplementarity ssDNA bridging with by its complementary ssDNA of sensing dissociate (Fig. 3).Thereby the g5p of polyhistidine tag will be fixed on and obtain directed g5p on the Ni-NTA bonded gold nano grain, and wherein this DNA binding site has activity by the peripheral direction towards this nano particle.The length of DNA and shape can influence the size or the form of this aggregate.The control of the g5p bonded nano particle of this DNA mediation can cause being used for the intelligent material Design of Sensor of DNA under biotic condition.
In some embodiments, the assembly of nano particle prepares by the following: with a plurality of nano particles of nucleotide binding protein functionalization, the nano particle of this functionalization is hatched with incomplementarity DNA and/or RNA, and at least two chains of this DNA and/or RNA are connected with nucleotide binding protein.By this way, the assembly that has prepared nano particle.
In order to obtain the accurate self-assembly of nano particle on multidimensional, the mixture of structurizing DNA and peptide-labeled g5p has been proposed, wherein this peptide can specificity be attached to nano particle.Can cause simple member to form the DNA support by the g5p tissue that is attached on the ssDNA zone, and affinity tag is used in further ordering processing nano particle (Fig. 5) on this DNA support.This method makes and might design complicated heterogeneous body nanostructure.
In some modification of multidimensional structure, the multidimensional structure of DNA and nano particle-functionalization g5p comprises the peptide that has the branched dna structure of a plurality of ssDNA, is attached to g5p, and specificity is attached to the nano particle of this peptide.In preferred embodiment, this g5p is attached on the ssDNA.By this way, form the composite structure of DNA and nano particle-functionalization g5p.
In some modification of multidimensional support, two other chains (chain 3 and 4) of the ssDNA that this multidimensional DNA support is included in two chains (chain 1 and 2) of the ssDNA that first locus interosculates by the C-ssDNA sequence, interosculate by the C-ssDNA sequence at second locus, the chain 2 and the chain 3 that interosculate by the C-ssDNA sequence at second locus, and one or more somes place is attached to g5p in the ssDNA chain at least two between the bonded locus.
In the preferred implementation of multidimensional support, this multidimensional support further comprises at least a nano particle at least one that is attached among the g5p.In the more preferably embodiment of multidimensional support, at least a nano particle is attached at least one zone of DNA.
In some embodiments, target is to improve the nanometer materials and the transmitter of DNA and protein induce.In some embodiments, this can realize like this: two antiparallel ssDNA are associated by g5p, and g5p is dissociated from ssDNA by the hybridization of complementary DNA or nucleic acid.
In some embodiments, the DNA assembly of g5p-mediation comprises incomplementarity DNA or the RNA chain that is attached on the nucleotide binding protein.By this way, formed assembly.
G5p is encoded by filobactivirus, thereby wherein it is attached to the precursor that ssDNA goes up formation phage particle assembly compoundly.External, g5p forms homodimer, and it in conjunction with two antiparallel ssDNA, induces the spiral rod shaped structure with 8～9nm external diameter with non-specificly.In conjunction with the monomeric Nucleotide number of each g5p is 2～4, and depends in conjunction with condition, comprises the ratio of protein and Nucleotide.The binding affinity of g5p and ssDNA, about 10 5To about 10 6M -1, depending on ssDNA sequence and salt concn, preferred combination is to structurizing DNA, as hair clip and G-four serobilas.Thereby utilized the assembling that is used for ssDNA-bonded nano particle to conciliate the proteic DNA binding characteristic of g5p of assembling.
For different binding characteristics and the preferred DNA topological framework thereof of studying g5p, designed exemplary dna structure: the double-stranded DNA of 19 base pairings (dsDNA) zone with three different zones, then be 32 Nucleotide antiparallels poly--3 ' the ssDNA afterbody that extends of T ssDNA zone and 15 Nucleotide.With g5p (in 10mM Tris-HCl, pH=7.4,200mM NaCl) this dna structure of titration (1 μ M), and utilize electrophoretic mobility to measure and studied the g5p combination.Observe three different g5p in conjunction with the stage, disclose its order in conjunction with (sequential binding) (Fig. 6 A).Because under high salt concn, the avidity of g5p and dsDNA is more much lower than the avidity of itself and ssDNA, so order of the occupy-place below inferring: be attached to antiparallel ssDNA zone, wherein the DNA band migration is bigger under 10 μ M g5p, then be attached to the ssDNA afterbody, be attached on the dsDNA during in g5p concentration at last greater than 35 μ M.Thereby g5p was attached to the ssDNA zone before being attached to the dsDNA zone, and it is higher than its binding affinity to the ssDNA afterbody slightly to the binding affinity in antiparallel zone.Associate to forming the influence of dsDNA heteroduplex in order to study g5p, and confirm that propose and the binding sequence ssDNA zone, adding can with equivalent (the 1 μ M) C-ssDNA of ssDNA afterbody hybridization.After adding C-ssDNA, observe two different band migration stages, wherein 20 μ M arrive different positions (Fig. 6 B) with 30 μ M g5p with higher mobility change, show that the fs of band migration is caused with combining of antiparallel ssDNA zone by g5p.Band tailing effect under 45 μ M g5p after the adding C-ssDNA shows that g5p is attached to dsDNA under this concentration.By relatively band under the 30 μ M g5p situations of existence and the intensity of not having the band under the g5p situation under controlled conditions, the hybridization efficiency of having determined C-ssDNA and g5p compound ssDNA afterbody is about 82%, shows the not remarkable effective DNA heterozygosis of inhibition of g5p.
In order to utilize the ability of g5p assembling ssDNA-bonded nano particle, synthesized ssDNA with about 50 copies (5 '-HS-C3H6-(T) 15-TAACCTAACCTTCAT-3 ') gold nano grain (Au) of (SEQ IDNO.1) parcel, and tested ssDNA-bonded gold nano grain (ssDNA-Au) assembly that g5p-mediates.Dynamic light scattering (DLS) is used for measuring hydromeehanics diameter value (D h) variation, it is relevant with geometry with aggregate size, particle interphase interaction.Fig. 7 A is illustrated in g5p and ssDNA-Au is hatched D during 20 minutes hInitial assembling speed extremely sensitive to g5p concentration, show the g5p dependency assembly of ssDNA-Au.Under the situation that does not have g5p, do not observe assembly.In order to study ssDNA-Au (20nM), utilize ultraviolet visible spectrophotometry (UV-vis) to study ssDNA-Au surface plasma (SP) resonance bands of a spectrum in the assembling of (～24 hours) between the incubation period of prolongation under a series of g5p concentration.These SP bands of a spectrum are related with the nanostructure of isolating Au and assembling.After increasing the g5p concentration that increases, isolating ssDNA-Au shows that at the SP at 525nm place bands of a spectrum red shift and band broadening the distance between the particle reduces or aggregate size increase (Fig. 7 B).Because the formation of big aggregate causes the solution turbidity to reduce, therefore more than 10 μ M g5p, observe higher delustring intensity (extinctionintensity).DLS and UV-vis result confirm that ssDNA-Au assembles by g5p.Utilize transmission electron microscope (TEM) to study the size and the form (Fig. 7 C) of the ssDNA-Au assembly of g5p-mediation.Find that this aggregate size increases along with the increase of g5p concentration, as disclosing by DLS and UV-vis, but not the numbers of particles of assembling reduces, and discloses the size of aggregate and can simply control by g5p concentration.The YieF protein (MW=20KDa) of polyhistidine tag is used as negative control, and it is not observed agglomeration of particles (Fig. 8 A and 8B) during DLS and UV-vis research.These results show that obviously g5p can be used for by changing the ssDNA-Au assembling of protein-nano particle ratio control.
In order to study the inhibition influence that dsDNA forms aggregate, before the assembling of beginning g5p-mediation, at first make particulate ssDNA-cap and C-ssDNA (5 '-ATGAAGGTTAGGTTA-3 ') (SEQ ID NO.2) part hybridize (Fig. 9 A).Utilize DLS to study of the variation (Fig. 9 B) of assembling speed to the C-ssDNA concentration-response.0.05 the time, this assembling speed sharply descends the mark (fc) of going up C-ssDNA and-ssDNA as Au greater than about.The ssDNA-Au assembling steric hindrance that this bigger inhibition influence announcement g5p-that is caused by low density dsDNA mediates is bigger.Incomplementarity ssDNA (NC-ssDNA, 5 '-AATATTGATAAGG ATAGC-3 ') (SEQ ID NO.3) be used as contrast, thus eliminate any inhibition titration effect that causes that combines by ssDNA in g5p and the solution.Find that NC-ssDNA is to the influence of assembling speed not significantly (Fig. 9 B) statistically.
The g5p that is incorporated into ssDNA can replace (Fig. 6 B) by C-ssDNA hybridization.This should cause dissociating of aggregate, and it can be used to detect the existence of C-ssDNA.In order to prove this point, prepared aggregate in about 24 hours by hatching ssDNA-Au (10nM) and g5p (5 μ M), after this, at room temperature hatched this aggregate and C-ssDNA about 12 hours.Utilize UV-vis monitoring colourity to change, its indication aggregate size changes.This peak position blue shift surpasses the C-ssDNA (fc=～0.4) (Figure 10 A) of nearly 200nM, indicates dissociating and the release of individual particle of little cluster (cluster).Observe, in the NC-ssDNA contrast, reduction is arranged this peak intensity slightly but (Figure 10 B) do not moved in the cutting edge of a knife or a sword position; NC-ssDNA makes aggregate be divided into littler cluster, but does not arrive the level of individual particle.TEM image with sample of 200nM C-ssDNA clearly illustrates dissociate (Figure 10 C) with discrete particles, and does not observe this dissociating for the sample with NC-ssDNA.
It is the useful property of g5p-ssDNA system that the aggregate of DNA control dissociates.In order to prove this point, by hatch the ssDNA-Au particle and with C-ssDNA (5 '-HS-C3H6-(T) 15-ATGAAGGTTAGGTTA-3 ') their the conventional hybridization method of target particle (C-ssDNA-Au) of (SEQ ID NO.4) parcel is assembled into aggregate with gold nano grain.After the formation, the conduct of dissociating of research aggregate increases the function of the C-ssDNA concentration that increases.Yet, near before about 16, do not observe aggregate dissociate (Figure 11) at fc.
Correctly assemble for ssDNA-Au and C-ssDNA-Au particulate, dna sequence dna inherent characteristic as G-C content, is controlled this assembling process usually.Use g5p as motivating force, the method for the present invention that is used for controlled incomplementarity ssDNA-bonded nanoparticle assemblies is novel.Assembling dissociating of dynamic (dynamical) control and particle aggregate can obtain by the sequence-specific hybridization with C-ssDNA, and the size of aggregate is controlled by regulating g5p concentration.
Another aspect of the present invention comprises molecular switch.In the modification of this molecular switch, it comprises " shutoff " position of quenching of fluorescence and " unlatching " position of fluorescent emission.In the preferred implementation of quenching of fluorescence " shutoff " position, thereby a ssDNA is attached at 5 ' end and forms fluorescein-ssDNA chain on the fluorescein, thereby the 2nd ssDNA is attached to DABCYL at 3 ' end and goes up formation ssDNA-DABCYL chain, and this fluorescein-ssDNA chain and this ssDNA-DABCYL chain and g5p combine.By making the hybridization of complementary ssDNA or ssRNA and fluorescein-ssDNA chain or ssDNA-DABCYL chain, can " open " this switch.By this way, removed the fluorescein quencher and made that fluorescence can be detected, this switch has been switched to " unlatching " position from " shutoff " position.
In the another kind of modification of this molecular switch, activated " unlatching " switch comprises first chain of dsDNA sections, ssDNA, and this first chain is on each end is attached in two chains of this dsDNA one and DABCYL.This switch also comprises second chain of ssDNA, and it is on each end is attached to another and fluorescein in two chains of this dsDNA, and two chains of this of ssDNA are not complementary each other.There are DABCYL and tip to have on the ssDNA chain of fluorescein by making g5p be attached at least one pair of tip, can " turn-off " this switch, make fluorescence pass through g5p quencher between the setting stage from fluorescein.
Peptide has been successfully used to make up the self-assembly of nanostructure and inorganic nanoparticles.The proteic genetically engineered of g5p that is used for show peptide provides the other chance that designs meticulous nano material.In the presence of C-ssDNA, need not the aggregate sensitivity that thermal treatment can carry out and separate assembling,, show that this approach is at design biological function cross materials with based on the potential aspect the biosensor of DNA as situation based on the aggregates of nanoparticles of complementary DNA; And these cross materialses of thermal treatment cause funtion part such as proteinic irreversible deactivation usually, and adding C-ssDNA should not influence their activity.We also notice and thisly can expand to the nano material of other type based on the new assembling approach of g5p and ssDNA, comprise carbon nanotube, semi-conductor and magnetic nanoparticle.
Fig. 1 illustrates and is used for the art methods that assembling is conciliate in the self-assembly of ssDNA-bonded nano particle.In this traditional method, the self-assembly of ssDNA-bonded nano particle is undertaken by making itself and complementary DNA hybridization, and separates assembling by carrying out more than the melting temperature (Tm) (Tm) that temperature is increased to hybrid dna.Nano particle 110 usefulness ssDNA sequences 112 functionalization, at least some of ssDNA sequence 112 are complimentary to one another.The complementary dna sequence 114 of nano particle allows hybridization 116, causes the self-assembly of nano particle.The nano particle 118 of assembling forms aggregate 120 then, and it can separate assembling by the solvent temperature that temperature is increased to dsDNA chain 126 more than 124, causes the depolymerization of functionalized nano particulate to conciliate assembling 128.
Fig. 2 illustrates the assembling of the incomplementarity ssDNA-bonded nano particle of controlled g5p-mediation and conciliates assembling.The inducing of inhibition reconciliation assembling that can observe assembling can be by causing with complementary DNA hybridization.Nano particle 210 usefulness incomplementarity DNA252 functionalization.Add g5p 254 and the nano particle self-assembly is joined in the assembly 256, and when needing, join in the aggregate 220.Add the depolymerization reconciliation assembling 260 that causes nano particle with the sequence complementary DNA of functionalized nano particle 258.
These assemblings/separating assembling process also can reversely carry out.Fig. 3 illustrates the g5p-bonded nano particle assembling of ssDNA mediation and conciliates the synoptic diagram of assembling.Here, nano particle 310 usefulness g5p 354 functionalization.Single stranded DNA 312 is joined in this solution and this functionalized nano particle self-assembly.When adding C-ssDNA 314, this nano particle is separated assembling.
Fig. 4 has described the functionalization of the molecular switch of g5p-mediation.G5p 454 makes DABCYL (Q) 464 move near fluorescein 466 with combining of antiparallel ssDNA zone 462, causes quenching of fluorescence.This can regard the molecular switch that is positioned at normal " shutoff " position as, or the manufacturing of detector.Fluorescent emission can be recovered by any one is hybridized.This process is equivalent to detect target or " unlatching " this molecular switch.
Fig. 5 illustrates the self-assembly of the nano particle of orderly peptide bonded g5p and DNA.Four branches with ssDNA are assembled by peptide bonded g5p 554 at the dna structure shown in this 570, and nano particle 510 is attached to this peptide and ordering by specificity.This can produce the nanoparticle structure of arbitrary structuresization.
Fig. 6 A and 6B illustrate the result that electrophoretic mobility is measured.Open up three regional titration dna fragmentations of benefit structure shown in Fig. 6 A with difference.This zone be 19 base pairs, antiparallel of dsDNA poly--the ssDNA afterbody of T ssDNA (32 bases) and 15 bases.The bonded model is depicted in the right group.The chart bottom illustrates has this trizonal structurizing DNA.Fig. 6 B has described the mobility of g5p mixture after C-ssDNA is attached to the ssDNA afterbody, and wherein the bonded model is in the group of the right side.
Fig. 7 A, 7B and 7C are the data from the ssDNA-bonded gold grain assembly of g5p-mediation.Figure among Fig. 7 A expresses, at ssDNA-Au (5nm) and g5p hydromeehanics diameter value (D between incubation period h) as the function of g5p (μ M) concentration, it utilizes the DLS analytical technology to carry out.Observe D hConcentration monotone increasing along with g5p.Fig. 7 B shows along with 10mMTris-HCl, and pH 7.44, and g5p concentration UV-vis spectrographic when 0 changes to 15 μ M changes in the 200mM NaCl solution.Along with the concentration increase of g5p, not only Strength Changes, and peak also moves (standing red shift) to longer wavelength.
Fig. 7 C is as 10mM Tris-HCl, and pH 7.44, in the 200mM NaCl solution g5p concentration when 0 is increased to 10 μ M, the TEM image of a series of nanoparticle assemblies form.Under the situation of no g5p, this nano particle is sparsely and relatively unevenly at interval on substrate.When concentration is increased to 2.5 μ M, form bigger, the nanoparticle assemblies of close interval more.The concentration of g5p increases further to that 5 μ M cause particle to begin to assemble.Being illustrated under the 10 μ M concentration nano particle virtually completely assembles.
Fig. 8 A and 8B illustrate the DLS and the UV-vis analytical results of ssDNA-bonded gold grain.In Fig. 8 A, DLS illustrates the D of the 5nmssDNA-Au of hatching with 2.5 μ M g5p and 2.5 μ M YieF hBe 5nm.Be illustrated in the D of ssDNA-Au under the existence of YieF hDo not change, and the D of the ssDNA-Au of hatching with g5p hIncrease to nearly 1.5 μ M.Fig. 8 B illustrates, and the UV-vis spectrum after 20nm ssDNA-Au and 15 μ M YieF are hatched nearly 20 hours illustrates peak position and is changed to about 525nm.
Fig. 9 A and 9B illustrate the synoptic diagram of the ssDNA-bonded gold grain assembly of g5p-mediation respectively, and the variation of the hydromeehanics diameter value of such assembly.Fig. 9 A schematically shows with dsDNA 952 and compares, and g5p 954 preferably combines with antiparallel ssDNA 912.Fig. 9 B illustrates per minute D hVariation as the function (the quantity mark of C-ssDNA in this solution, that is, C-ssDNA quantity is divided by the total quantity of ssDNA on this gold nano grain 910) of fc.This illustrates, at ssDNA-bonded gold grain (ssDNA-Au ≈ 5nM) the assembly process D of (2.5 μ M) g5p albumen-mediation hVariation, wherein this gold grain and C-ssDNA prehybridization.
Figure 10 A, 10B and 10C have described, the decomposition of the ssDNA-bonded gold grain aggregate of g5p-mediation when adding C-ssDNA.Figure 10 A and 10B are illustrated in C-ssDNA (Figure 10 A) or NC-ssDNA (Figure 10 B) and hatch the about 12 hours UV-vis data of aggregates of nanoparticles (10nMssDNA-Au and 5 μ M g5p) afterwards.As can be seen from the figure, the peak absorbing wavelength of the ssDNA-Au mixture of hatching with C-ssDNA is moved, and those of hatching with NC-ssDNA then do not have.Figure 10 C is illustrated in 100nM (fc=～0.2) and 200nM C-ssDNA (fc=～0.4) and hatches the TEM image of sample afterwards.
Figure 11 illustrates and adds the influence of C-ssDNA to the gold grain aggregate.The conventional hybridization method of the target particle (C-ssDNA-Au) of this complementation of the usefulness ssDNA parcel by hatching ssDNA-Au and they is assembled into aggregate with gold nano grain.Single stranded DNA-Au (10nM) and C-ssDNA-Au (10nM) are at 10nM Tris-HCl, and pH 7.4, hatch among the 200mM NaCl about 24 hours, make the peak move to about 548nm (0 μ M 1132) from about 525nm (contrast 1130).After hatching, do not observe the recovery that the peak moves with C-ssDNA (8 μ M 1134).
Illustrative methods
Following clearly description only provides as specific examples.The content that this paper comprises does not hint the endorsement of supporting any product or manufacturers.Concrete trade(brand)name, model and manufacturers only provide for specifying, and the reagent of the equipment of available similar functions and similar quality replaces.In addition, all observed values of time, quantity, concentration etc. are all in experiment and personal errors scope.
The protein preparation
The g5p gene of phage M13 (New England Biolabs (NEB)) utilize primer 5 '-TAATTCC ATATGATTAAAGTTG AAATTA AACC A-3 ' (SEQ ID NO.5) and 5 '-TAGCTTGCTCTTCCGCACTTAGCCGGAACGAGGCG-3 ' (SEQ ID NO.6) increases by polymerase chain reaction (PCR).This causes the side that the dna fragmentation of NdeI and SapI restriction site is arranged usually.This PCR product digests with NdeI and SapI (NEB) and is connected in the pET-30b carrier (Novagen).After confirming sequence, utilize Gene Pulser XcellSystem (Bio-Rad) that recombinant plasmid is incorporated in the BL21-DE3 electricity transformed competence colibacillus cell.By (LB) adding sec.-propyl-β-D-1-sulfo-breast pyranoside (IPTG) in the substratum (Sigma) having kantlex (Kanamycin) (100 μ g/ml) bacteriolyze meat soup (lysogeny broth) (sigma), induce the G5p protein expression.After the expression phase, use the ultrasonication cell, and utilize the g5p albumen of Ni-NTA post (Qiagen) purifying His mark.Utilize fast protein liquid chromatogram (FPLC) (AKTA explorer, GE Healthcare) to obtain other depositing of g5p, this chromatogram has propylene dextrane gel S-200 high resolving power screening (sizing) post (Amersham Biosciences).Obtain＞95% purity, as after on the 15%SDS-polyacrylamide gel, dyeing, by utilizing Quantity One software (Bio-Rad) analysing protein bands of a spectrum determined with Coomassie blue.7450M -1Cm -1Molar extinction coefficient be used for determining protein concn.According to Zhang, Y.B. wait people Functionalized carbon nanotubesfor detecting viral proteins.Nano Letters, 7 (10): pp.3086-3091,2007 previously described this YieF protein that prepare are incorporated herein its full content with way of reference.
Electrophoretic mobility is measured
By use respectively sequence 5 '-GACCACATACCGCACCATC (T) 32CTGCTACGAGACTTC-3 ' (SEQ ID NO.7) and 5 '-(T) 32GATGGTGCGGTATGTGGTC-3 ' (SEQ ID NO.8) two ssDNA that anneal obtain to be used for the DNA in conjunction with research.Under 37 ℃, hatched this annealed DNA (1 μ M) and g5p 10 minutes.By ethidium bromide staining visual this DNA after sepharose (2.5%) electrophoresis.To have sequence 5 '-C-ssDNA of GAA GTC TCGTAG CAG-3 ' (SEQ ID NO.9) joins in the mixture of g5p and annealed dna, and hatching under 37 ℃ 15 minutes before the electrophoresis.After ethidium bromide staining, analyze the relative intensity of these DNA bands of a spectrum.
Feature
Utilize PerkinElmer Lambda 35 spectrographs to obtain uv-vis spectra.Utilize MalvernZetasizer ZS apparatus measures DLS, this instrument is equipped with the backscatter detector of 633nm laser and 173 °.For visual ssDNA-Au, operation JEOL 1300TEM under 120kV.By hatching sample 10 minutes on the copper grid that applies at carbon and clean twice, preparation TEM sample with distilled water.
Consider the instruction of this paper, the inventor and others skilled in the art can be extended for this method the assemble nanometer particulate more in complex DNA-g5p structure.The demonstration of the functional peptide of the C-end of g5p can allow the nano material of biologically active.In addition, for more potential application, consider to use the nano particle (semi-conductive, magnetic) of other classification.The operation described (work) can be further used for developing biosensor, comprises that exploitation is used for the approach of microarray design.
Present method makes and need not thermal treatment can be controlled DNA-bonded nano particle under biotic condition assembling reconciliation assembling, in the feasible nanostructure that easily protein is incorporated into based on DNA, the very big potentiality of hint design composite nano materials, and can be designed for and detect nucleotide sequence (DNA RNA) need not they are carried out the approach of mark.
Though carried out the description of front with reference to single embodiment of the present invention, should be appreciated that the instruction that utilizes this paper, those skilled in the art can propose various variations and modification aspect more and not depart from the present invention.For example, can utilize gene protein except that g5p.In another embodiment, the present invention can be used to detect the chemical substance that is used for Secure Application.
The description of front is illustrative, and the present invention is only defined by the appended claims.
Figure IDA0000046020080000011
Figure IDA0000046020080000021

Claims (42)

1. a controlled and reversible nanoparticle assemblies comprise the nano particle with incomplementarity DNA and/or RNA parcel, thereby described incomplementarity DNA and/or RNA forms nanoparticle assemblies by the nucleotide binding protein combination.
2. controlled and reversible nanoparticle assemblies according to claim 1 wherein, by further making described nanoparticle assemblies and combining with described incomplementarity DNA and/or RNA complementary DNA and/or RNA, makes described nanoparticle assemblies separate assembling.
3. controlled and reversible nanoparticle assemblies according to claim 1, wherein, described nano particle is a metal.
4. controlled and reversible nanoparticle assemblies according to claim 3, wherein, described metal is selected from the group of being made up of gold and silver and platinum.
5. controlled and reversible nanoparticle assemblies according to claim 4, wherein, described metal is a gold.
6. controlled and reversible nanoparticle assemblies according to claim 1, wherein, described nano particle is a semi-conductor.
7. controlled and reversible nanoparticle assemblies according to claim 6, wherein, described semi-conductor is selected from the group of being made up of cadmium selenide, Cadmium Sulfide, zinc sulphide and gallium arsenide.
8. controlled and reversible nanoparticle assemblies according to claim 1, wherein, described nano particle is a magnetic.
9. controlled and reversible nanoparticle assemblies according to claim 8, wherein, described nano particle is a ferriferous oxide.
10. controlled and reversible nanoparticle assemblies according to claim 1, wherein, described DNA and/or RNA are strands.
11. controlled and reversible nanoparticle assemblies according to claim 1, wherein, described DNA and/or RNA are double-stranded.
12. controlled and reversible nanoparticle assemblies according to claim 1, wherein, described nucleotide binding protein is g5p.
13. the method for assembling is conciliate in a controllable nano particle assembling, comprising:
With DNA and/or RNA parcel nano particle;
The nano particle of described parcel is combined with nucleotide binding protein; And
Described DNA and/or RNA are attached on the described nucleotide binding protein, thereby form the assembly of nano particle.
14. the method for assembling is conciliate in controllable nano particle assembling according to claim 13, wherein, by further making described nanoparticle assemblies and combining, make the assembly of described nano particle separate assembling with described incomplementarity DNA and/or RNA complementary Nucleotide.
15. the method for assembling is conciliate in controllable nano particle assembling according to claim 13, wherein, described nano particle is a metal.
16. the method for assembling is conciliate in controllable nano particle assembling according to claim 15, wherein, described metal is selected from the group of being made up of gold and silver and platinum.
17. the method for assembling is conciliate in controllable nano particle assembling according to claim 16, wherein, described nano particle is a gold.
18. the method for assembling is conciliate in controllable nano particle assembling according to claim 13, wherein, described nano particle is a semi-conductor.
19. the method for assembling is conciliate in controllable nano particle assembling according to claim 18, wherein, described semi-conductor is selected from the group of being made up of cadmium selenide, Cadmium Sulfide, zinc sulphide and gallium arsenide.
20. the method for assembling is conciliate in controllable nano particle assembling according to claim 13, wherein, described nano particle is a magnetic.
21. the method for assembling is conciliate in controllable nano particle assembling according to claim 20, wherein, described nano particle is a ferriferous oxide.
22. the method for assembling is conciliate in controllable nano particle assembling according to claim 13, wherein, described DNA and/or RNA are strands.
23. the method for assembling is conciliate in controllable nano particle assembling according to claim 13, wherein, described DNA and/or RNA are double-stranded.
24. the method for assembling is conciliate in controllable nano particle assembling according to claim 13, wherein, described nucleotide binding protein is g5p.
25. one kind is used for assemble nanometer particulate method, comprises:
With a plurality of nano particles of nucleotide binding protein functionalization;
The nano particle of described functionalization is hatched with incomplementarity DNA and/or RNA; And
At least two chains of described DNA and/or RNA are connected with described nucleotide binding protein, thereby form the assembly of nano particle.
26. method according to claim 25, wherein, described nano particle is a metal.
27. method according to claim 26, wherein, described metal is selected from the group of being made up of gold and silver and platinum.
28. method according to claim 27, wherein, described nano particle is a gold.
29. method according to claim 25, wherein, described nano particle is a semi-conductor.
30. method according to claim 29, wherein said semi-conductor is selected from the group of being made up of cadmium selenide, Cadmium Sulfide, zinc sulphide and gallium arsenide.
31. method according to claim 25, wherein, described nano particle is a magnetic.
32. method according to claim 31, wherein, described nano particle is a ferriferous oxide.
33. the molecular switch of the open site of scram position that comprises quenching of fluorescence and fluorescent emission, wherein,
Thereby be attached on the fluorescein ssDNA that forms fluorescein-ssDNA chain, be attached to DABCYL at 3 ' end and go up the 2nd ssDNA that forms the ssDNA-DABCYL chain thereby the scram position of described quenching of fluorescence is included in 5 ' end, described fluorescein-ssDNA chain and described ssDNA-DABCYL chain and g5p combine; And
The open site of described fluorescent emission further comprises complementary ssDNA or the ssRNA with described fluorescein-ssDNA chain or the hybridization of described ssDNA-DABCYL chain, thereby removes the fluorescein quencher and can realize fluoroscopic examination.
34. a molecular switch comprises:
The sections of dsDNA;
Article one chain of ssDNA, it is on each end is attached in two chains of described dsDNA one and DABCYL;
The second chain of ssDNA, it is on each end is attached to another and fluorescein in two chains of described dsDNA, and two chains of described ssDNA are not complementary each other; And
G5p, being attached at least one pair of tip has DABCYL and tip to have on the ssDNA chain of fluorescein, so that passing through g5p quencher between the setting stage from the fluorescence of described fluorescein.
35. the DNA assembly of a g5p mediation comprises incomplementarity DNA or RNA chain, described incomplementarity DNA or RNA link are incorporated on the nucleotide binding protein, thereby form assembly.
36. the DNA assembly of g5p mediation according to claim 35, wherein, described DNA or RNA are strands.
37. the DNA assembly of g5p mediation according to claim 35, wherein, described nucleotide binding protein is g5p.
38. one kind is used for assemble nanometer particulate method, described method comprises:
With a plurality of nano particles of incomplementarity ssDNA functionalization;
The nano particle of described functionalization is exposed to gene 5 albumen; And
At least two chains of described ssDNA are linked to each other with g5p, thereby form the assembly of nano particle.
39. the multidimensional structure of the g5p of a DNA and nano particle-functionalization, comprise the peptide that has the branched dna structure of a plurality of ssDNA, is bonded to g5p, wherein said g5p is attached on the described ssDNA, and nano particle is incorporated into described peptide specifically, thereby forms the composite structure of DNA and nano particle functionalization g5p.
40. a multidimensional DNA support comprises:
Article one chain and the second chain of the ssDNA that interosculates by the C-ssDNA sequence at first locus;
The 3rd chain and the 4th chain of the ssDNA that interosculates by the C-ssDNA sequence at second locus;
Described second chain and the 3rd chain of the ssDNA that interosculates by the C-ssDNA sequence at second locus; And
Be attached to the g5p at least two chains of ssDNA at the one or more somes place between the described bonded locus.
41., further comprise at least a nano particle that is incorporated at least one described g5p according to the described support of claim 40.
42., further comprise at least a nano particle that is incorporated at least one DNA zone according to the described support of claim 41.
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